The present invention pertains to a mammalian DNA mismatch repair gene, MSH5, and uses thereof, for example, in drug screening, cancer prognosis and diagnosis. The gene product is required for meiotic crossing over and segregation of chromosomes during meiosis. More specifically, the invention relates to detection of alterations in the gene which are associated with some mammalian, preferably human, cancers, as well as conditions involving problems in meiotic segregation.
Accurate transmission of genetic information is important in the survival of a cell, an organism, and a species. A number of mechanisms have evolved that help to ensure high fidelity transmission of genetic material from one generation to the next since mutations can lead to new genotypes that may be deleterious to the cell. DNA lesions that frequently lead to mutations are modified, missing or mismatched nucleotides. Multiple enzymatic pathways have been described in prokaryotic systems that can specifically repair these lesions.
There are at least three ways in which mismatched nucleotides arise in DNA. First, physical damage to the DNA or DNA precursors can give rise to mismatched bases in DNA. For example, the deamination of 5-methyl-cytosine creates a thymine and, therefore, a G-T mispair. Second, misincorporation, insertion, or deletion of nucleotides during DNA replication can yield mismatched base pairs. Finally, genetic recombination produces regions of heteroduplex DNA which may contain mismatched nucleotides when such heteroduplexes result from the pairing of two different parental DNA sequences. Mismatched nucleotides produced by each of these mechanisms are known to be repaired by specific enzyme systems.
The well defined mismatch repair pathway is the E. coli MutHLS pathway that promotes a long-patch (approximately 3 Kb) excision repair reaction which is dependent on the mutH, mutL, mutS and MutU(uvrD) gene products. The MutHLS pathway appears to be the most active mismatch repair pathway in E. coli and is known to both increase the fidelity of DNA replication and act on recombination intermediates containing mispaired bases. This system has been reconstituted in vitro and requires the MutH, MutL, MutS and UvrD (helicase II) proteins along with DNA polymerase III holoenzyme, DNA ligase, single-stranded DNA binding protein (SSB) and one of the single-stranded DNA exonucleases, Exo I, Exo VII or RecJ. MutS protein binds to the mismatched nucleotides in DNA. MutH protein interacts with GATC sites in DNA that are hemi-methylated on the A and is responsible for incision on the unmethylated strand. Specific excision of the unmethylated strand results in increased fidelity of replication because excision is targeted to the newly replicated unmethylated DNA strand. MutL facilitates the interaction between MutS bound to the mismatch and MutH bound to the hemi-methylated Dam site resulting in the activation of MutH. UvrD is the helicase that appears to act in conjunction with one of the single-stranded DNA specific exonucleases to excise the unmethylated strand leaving a gap which is repaired by the action of DNA polymerase III holoenzyme, SSB and DNA ligase. In addition, E. coli contains several short patch repair pathways including the VSP system and the MutY (MicA) system that act on specific single base mispairs.
In bacteria, therefore, mismatch repair plays a role in maintaining the genetic stability of DNA. The bacterial MutHLS system has been found to prevent genetic recombination between the divergent DNA sequences of related species such as E. coli and S. typhimurium (termed: homologous recombination).
A number of human mismatch repair genes have been discovered. Defects in the human MSH2 gene are associated with Hereditary Non-Polyposis Colon Cancer (HNPCC), a familiar form of human colorectal cancer (CRC) that is also known as Lynch""s Syndrome. Other mismatch repair genes discovered in humans include MLH 1.
These genes are not only involved with susceptibility to cancer, but can be associated with other aspects. For example, defects in MSH2 and MLH1 confer resistance to alkylating agents frequently used in treating cancers. Consequently, the discovery of mismatch repair genes is extremely important. For example, finding a new mismatch repair gene permits one to look for defects in that gene and determine its association with particular cancers. This not only permits one to determine susceptibility to particular cancers, but to have a better prognosis of the disease and to more fully understand what therapies to use. Thus, being able to fmd additional mammalian, particularly human, mismatch repair genes is very important.
We have discovered and sequenced mammalian MSH5 genes which are involved in the DNA mismatch repair pathway. We have identified its chromosomal location in humans as well as the intron-exon borders in both mice and humans. This gene produces a protein involved in meiotic crossing over and segregation of chromosomes during meiosis. Thus, defects in the gene should indicate susceptibility to disorders associated with those activities such as Downs Syndrome and certain types of infertility. Further defects in mismatch repair genes indicate susceptibility to various types of cancer. Moreover, defects in this gene confer resistance to alkylating agents. Alkylating agents represent a preferred class of chemotherapeutic agents frequently used in treating cancer.
Consequently, individuals diagnosed with cancer should have that cancer screened for the presence of a defect in the MSH5 gene. If the individual has such a defect, then an agent other than an alkylating agent should be prescribed. This gene, also has other applications. It can be used in assays, to express gene product, for drug screens, and therapeutically.
SEQ ID NO.:1 is the nucleotide sequence of the human MSH5 MSH2 gene.
SEQ ID NO.:2 is the deduced amino acid sequence of the human MSH5 gene product.
SEQ ID NOs.:3-26 are the nucleotide sequence of the 5=exon-intron borders.
SEQ ID NOs:27-50 are the nucleotide sequences of the 3=intron-exon borders.
SEQ ID NOs: 51 and 52 are primers used in screening for human genomic MSH5.
SEQ ID NO:53 is the nucleotide sequence of the murine MSH5 gene.
SEQ ID NO:54 is the deduced amino acid sequence of the murine MSH5 gene product.
SEQ ID NOs:55-85 represent nucleotide intronic sequences of human MSH5.
SEQ ID NOs:86-90 are nucleotide sequences of the 5xe2x80x2 exon-intron borders of mMSH5.
SEQ ID NOs:91-95 are nucleotide sequences of the 3xe2x80x2 intron-exon borders of mMSH5.
SEQ ID NOs:96-100 represent nucleotide intronic sequences of murine MSH5.
SEQ ID NOs:101-104 are primers used.